Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms

ABSTRACT

The verified cDNA sequences for human, bovine and porcine lactoferrin protein have been used to prepare recombinant lactoferrin for therapeutic and nutritional applications. Regions of the cDNA such as the Fe binding sites can be used to make an hLF polypeptide product The present invention provides novel plasmids, transfected eucaryotic cells and methods of producing these plasmids and transfected eucaryotic cells. The novel plasmid contains the cDNA for lactoferrin protein. Methods for the production of lactoferrin protein in fungi and bacteria are also provided. Thus, the present invention provides an efficient and economical means for the production of recombinant lactoferrin protein and lactoferrin related polypeptides.

RELATED APPLICATIONS

This application is a continuation in part of pending application Ser.No. 07/967,947, filed Oct. 27, 1992, which in turn is a continuation ofapplication Ser. No. 07/348,270, filed May 05, 1989, now abandoned. Thisapplication is also a continuation in part of pending application Ser.No. 07/873,304 filed Apr. 24, 1992.

This invention was made with government support under Grant No. HD27965awarded by the National Institute of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of iron-bindingglycoproteins. More specifically, the present invention relates to therecombinant production of various lactoferrins.

2. Description of the Prior Art

Lactoferrin (LF) is an iron-binding glycoprotein found in milk and othersecretions and body fluids. It is one of a number of iron bindingproteins, sometimes referred to as transferring, and is involved in ironbinding and delivery in mammals.

Human lactoferrin (hLF) is a member of the transferrin family ofiron-binding monomeric glycoproteins. It was originally discovered inmilk where it can reach levels of 7 grams/liter in colostrum. LF hassince been detected in other external fluids of humans and othermammals. The fluids include tears, saliva and mucosal secretions andalso in the secondary granules of polymorphonuclear leukocytes.

Lactoferrin has been implicated as a factor in resistance againstenteritis infections in suckled newborn humans. Thebacteriocidal/bacteriostatic actions are considered to be due at leastin part to the iron binding properties of lactoferrin. Lactoferrindecreases the iron availability to iron-requiring microorganisms andthereby interferes with their growth and reproduction. At least onenon-ironbinding bactericidal domain has also been reported for humanlactoferrin. Lactoferrin is also considered to have antiviral propertiesand to have other potential therapeutic applications.

LF is a 78 kilo Dalion (k Da) glycoprotein having a bilobal structurewith a high degree of homology between the C and N terminal halves whichis evident at both the amino acid and three dimensional structurallevel. Each of these lobes can reversibly bind one ferric iron with highaffinity and with the concomitant binding of bicarbonate. The biologicalfunctions proposed for lactoferrin include protection against microbialinfection, enhanced intestinal iron absorption in infants, promotion ofcell growth, regulation of myelopoiesis and modulation of inflammatoryresponses.

Human lactoferrin (hLF) has a high affinity for iron and two Fe³⁺cations can be bound per molecule. The complete HLF protein has beensubjected to amino acid sequencing and is reported to have 703 aminoacids. There are two glycosylation sites. Metz-Boutigue et al., Eur. JBiochem., 145:659-676 (1984). Anderson et al., Proc. Nat'l Acad. Sci.USA, 84:1769-1773 (April 1987).

In other studies, a cloned cDNA probe for amino acids 428 to 703 of theMetz-Boutigue structure of the lactoferrin protein was isolated. ThecDNA sequence was in general agreement with the earlier analysis of theamino acid sequence of the protein. Rado et al., Blood, 79; 4:989-993,79; 4:989-993 (October 1987). The probe was reported to encompassapproximately 40% of the coding region and the 3′ terminus. The cDNAsequence for both porcine, Lydon, J. P., et al., Biochem. Biophysic.ACTA, 1132:97-99 (1992); Alexander, L. J., et al., Animal Genetics,23:251-256 (1992) and bovine lactoferrin, Mead, P. E., et al., NucleicAcids Research, 18:7167 (1990); Pierce, A., et al., Eur. J Biochem.,196:177-184 (1991), have been determined.

Polypeptides derived from lactoferrin are also known to be biologicallyactive. A fragment containing a possible iron binding site was reportedby Rado, et al. supra. An N-terminal human lactoferrin fragment,including a bactericidal domain of HLF, was isolated from a pepsindigest. Bellamy, W. M., et al, Biochem. Biophys. ACTA, 1121:130-136(1992). Synthetic 23 and 25 amino acid polypeptides were synthesized andfound to have activities similar to the fragments derived by pepsindigestion. The synthesis details, yields and purity of the syntheticpeptides were not reported. Bellamy et al. do not provide a practicalroute to large scale production of the polypeptides free of thecontaminates resulting form isolation from natural products.

The bactericidal domain from lactoferrin has a broad spectrum ofantimicrobial action. Bellamy, W. M. et al., J. App. Bact. 73, 472-479(1992). Although Bellamy et al. report that bovine lactoferrin isolatedfrom milk can provide commercial quantities of the bovine polypeptide bypepsin digestion, the materials used in both studies had a minimumpurity of only 95%. Bellamy, et al. do not provide constructs for thelarge scale production of synthetic human or bovine lactoferrin orlactoferrin polypeptides. Neither does Bellamy et al. provide theability to produce peptides that are not available by enzyme digestion.

Filamentous fungi have been successfully employed as hosts in theindustrial production of extracellular glycoproteins. Certain industrialstrains are capable of secreting gram quantities of these proteins. Inaddition, filamentous fungi are able to correctly performpost-translational modifications of eucaryotic proteins and many strainshave U.S. Food and Drug Administration approval. Furthermore, largescale fermentation technology and downstream processing experience isavailable.

Currently, there is no efficient and economical way to produce hLF,other species lactoferrin, or to control production of lactoferrinpolypeptides. Consequently, a long felt need and description in this artwould be met by the development of an efficient method for theproduction of human lactoferrin for nutritional and therapeuticapplications and for further investigation into its mechanism of action.

SUMMARY OF THE INVENTION

The invention comprises the verified cDNA sequences for humanlactoferrin, and cDNA expression systems for use of various lactoferrinDNA sequences to produce human, bovine, porcine and other lactoferrinsfor a variety of end uses. The cDNA expression systems of the inventionalso provide a practical route and method to make lactoferrinpolypeptides or fragments having biological activity. The hLF cDNAincludes an open reading frame of 2133 nucleotides coding for a proteinof 711 amino acids. These 711 amino acids include 19 amino acidscorresponding to a secretion signal peptide sequence followed by 692amino acids of mature human lactoferrin. The cDNA sequence and deducedamino acid sequence differ from the previously published data ofMetz-Boutigue, supra.

In one embodiment, the present invention provides for a recombinantplasmid comprising the cDNA of human or other lactoferrin. The plasmidof the present invention is adapted for expression in a eucaryotic celland contains the regulatory elements necessary for the expression of thehuman lactoferrin cDNA in this eucaryotic cell.

In another embodiment, the present invention provides for a transformedcell which includes a heterologous DNA sequence which codes forlactoferrin or a polypeptide related to lactoferrin. The heterologousDNA sequence will preferably be incorporated into a plasmid. Eucaryotichost cells are selected from the group consisting of mammalian cells,immortalized mammalian cells, fungi or yeasts. Preferred cells includefilamentous fungi comprising Aspergillus, and yeasts. The plasmidcontains a plasmid vector into which a polydeoxyribonucleotide (DNA)segment coding for human or other lactoferrin protein has been inserted.

In yet another embodiment of the present invention, there is provided aprocess for producing recombinant human or other lactoferrin whichcomprises culturing a transformant eucaryotic cell, which includes arecombinant plasmid. The plasmid contains a plasmid vector having apolydeoxyribonucleotide coding for the lactoferrin protein. Afterculturing in a suitable nutrient medium until lactoferrin protein isformed, the lactoferrin protein is isolated.

In still yet another embodiment of the present invention, there isprovided a recombinant expression vector. This vector comprises atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression; (2) cDNA codingfor lactoferrin; (3) appropriate transcription and translationinitiation and termination sequences; and (4) a genetic element forselection of transformed cells or spores such as Aspergillus spores thathave been transformed with the vector.

In still yet another embodiment of the present invention, there isprovided a method for producing biologically active recombinantlactoferrin. The method comprises synthesizing sequences containing aselectable marker gene, a promotor, a transcription terminationsequence, and a linker sequence; cloning the sequences to form aplasmid; digesting the plasmid with a restriction endonuclease;inserting a cDNA coding for lactoferrin into a restriction site; andtransforming eucaryotic cells with the plasmid expressing lactoferrincDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features, advantages, andobjects of the invention, as well as others which will become clear, areobtained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of this specification.

It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore not to beconsidered limiting of its scope. The invention may admit to otherequally effective equivalent embodiments.

FIG. 1 is a schematic drawing of the hLF cDNA including the locations ofthe 5′ untranslated region, the secretion peptide signal sequence,mature lactoferrin and 3′ untranslated region.

FIG. 2 is the cDNA sequence (SEQ. ID No. 1) with deduced amino acids(SEQ. ID No. 2) for the human lactoferrin protein and signal peptidesequence.

FIG. 3 is a schematic representation of an autoradiograph of recombinanthuman lactoferrin protein expressed from the complete cDNA.

FIG. 4 is a schematic representation of an autoradiograph of the resultsof in vitro translation of a 2,140 bp human lactoferrin sequence and hLFprotein in reticulocyte lysates.

FIG. 5 depicts a schematic representation of the Aspergillus oryzaeexpression plasmid, pAhlfg.

FIG. 6 shows a southern blot analysis of transformed Aspergillus oryzaestrains.

FIG. 7 depicts an RNA analysis of transformant versus control A07.

FIG. 8 shows the silver stained SDS-acrylimide gel analysis ofrecombinant LF secretion and purification.

FIG. 9 illustrates the characterization of recombinant human LF.

FIG. 10 is a western immunoblot of cellular extracts of transformedE.coli cells expressing the C terminal fragment of LF.

FIG. 11 shows the coomassie-stained SDS-PAGE analysis of extracts oftransformed E. coli cells expressing the C terminal fragment of LF.

FIG. 12 shows the expression and purification of the glutathioneS-transferase/LFN-1 fusion protein.

FIG. 13 Schematic representation of the A. Oryzae universal expressionplasmid, pAG.

FIG. 14 is the (A) cDNA sequence (SEQ. ID No. 3) with (B) deduced aminoacids (SEQ. ID No. 4) for the bovine lactoferrin protein.

FIG. 15 is the (A) cDNA sequence (SEQ. ID No. 5) with (B) deduced aminoacids (SEQ. ID No. 6) for the porcine lactoferrin protein.

FIG. 16 is a Western blot showing hLF expression in SaccharomycesCervisiae.

FIG. 17 is a schematic of the plasmid used for expression of the cDNA(SEQ. ID No. 1) in Aspergillis Nidulans.

FIG. 18 shows restriction enzyme cleavage sites for the human cDNAsequence.

FIG. 19 shows restriction enzyme cleavage sites for the bovine cDNAsequence.

FIG. 20 shows restriction enzyme cleavage sites for the porcine cDNAsequence.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

For the purposes of the present application, the term “transferrinfamily” means a family of iron transferring proteins including serumtransferrin, ovotransferrin and lactoferrin. These proteins are allstructurally related.

For the purposes of the present application, the term “vector(s)” meansplasmid, cosmid, phage or other vehicle to allow insertion, propagationand expression of lactoferrin cDNA.

For the purposes of the present application, the term “host(s)” meansany cell that will allow lactoferrin expression.

For the purposes of the present application, the term “promotor(s)”means regulatory DNA sequences that controls transcription of thelactoferrin cDNA.

For the purposes of the present application, the term “multiple cloningcassette” means a DNA fragment containing restriction enzyme cleavagesites for a variety of enzymes allowing insertion of a variety of cDNAs.

For the purposes of the present application, the term “transformation”means incorporation permitting expression of heterologous DNA sequencesby a cell.

For the purposes of the present application, the term “iron bindingcapacity” means ability to bind Fe. Fully functional human lactoferrincan bind two atoms of iron per molecule of LF.

For the purposes of the present application, the term “biologicalactivity/biological active” means biological activity of lactoferrin asmeasured by its ability to bind iron, or kill microorganisms, or retardthe growth of microorganisms, or to function as an iron transferprotein.

For the purposes of the present application, the term “substitutionanalog” referring to a DNA sequence means a DNA sequence in which one ormore codons specifying one or more amino acids of lactoferrin or alactoferrin polypeptide are replaced by alternate codons that specifythe same amino acid sequence with a different DNA sequence. Where“substitution analog” refers to a protein or polypeptide it means thesubstitution of a small number, generally five or less, commonly 3 or 4,and more often 1 or 2 amino acids as are known to occur in allelicvariation in human and other mammalian proteins wherein the biologicalactivity of the protein is maintained. For example, hLF isolated frommilk has been reported to differ from the hLF of SEQ. ID No. 2 at twoamino acid residues.

The confirmation of the cDNA sequence and the deduced amino acid havebeen proven by multiple confirmation procedures. These are:

-   -   1. Multiple sequence analyses.    -   2. Comparison of the amino acid sequence deduced from the cDNA        with that of hLF generated by conventional amino acid sequencing        of hLF isolated from milk. The unique cDNA sequence which        encodes the human lactoferrin protein has a variety of        applications as known and indicated in the literature.    -   3. Transcription and translation of hLF protein from the cDNA        with positive identification using an anti-hLF antibody.

The cDNA sequence of the present invention can be used to preparerecombinant human lactoferrin, thus making available a source of proteinfor therapeutic and nutritional applications. The confirmed cDNA of thisinvention can be used in an appropriate cloning vehicle to replicate thecDNA sequence. Also, the cDNA can be incorporated into a vector systemfor human lactoferrin expression. Other lactoferrin DNA sequences can besubstituted for the human lactoferrin cDNA sequence to provide bovine,porcine, equine or other lactoferrins. Partial cDNA sequences can alsobe employed to give desired lactoferrin derived polypeptides. Theexpression systems of the invention can be used to provide lactoferrinderived polypeptides that are not available by enzymatic digestion ofnaturally occurring lactoferrin. The invention further provides anexpression system for producing lactoferrin and lactoferrin relatedpolypeptides in mammalian cell lines, other eucaryotic cells includingyeast and fungal cells and procaryotic cells. The invention allows forthe production of lactoferrin free of lactoperoxidase, lysozyme, orother proteins that are contaminants of lactoferrin isolated from milkor other natural products. This invention is not limited to anyparticular uses of the human cDNA sequence or production of lactoferrinof other species from the appropriate DNA sequences.

The recombinant LF being a protein derived by recombinant techniques canbe used in a variety of applications. The human gene can be transferredto mammalian systems such as cows and other agriculturally importantanimals and expressed in milk. The incorporation of a human lactoferringene and expression in the milk of animals can combat an iron deficiencytypical in piglets. The inclusion of the human lactoferrin gene withexpression should improve an animal's disease resistance to bacterialand viral infection. The tissue specific expression of human lactoferrinin mammary glands, for instance, would impart the bacteriocidal andvirucidal benefit of the expressed gene to young feeding on the milk andwould provide a production means for the secreted protein fortherapeutic use.

The gene can be placed in the appropriate cloning vector for theproduction of LF. The LF produced by recombinant methods can be used ina variety of products including human or animal foods, as therapeuticadditives to enhance iron transport and delivery, and for the virucidaland bacteriocidal qualities, as additives for eyedrops, contact lens andother eye care solutions, topical skin care products, eardrops,mouthwashes, chewing gum and toothpaste. The recombinant LF wouldprovide a safe, naturally occurring product which can be topicallyapplied as well as ingested safely. The bactericidal lactoferrinpolypeptides are useful as preservatives in the above listed products,and as therapeutic anti-infection agents. The iron binding polypeptidesare useful as iron carrier proteins for nutritional and therapeuticuses, and as bacteriostats and bactericides, especially in products ofthe types listed above. Each protein may also be used as a nutritionsupplement and as a source of amino acids.

The full-length cDNA encoding human lactoferrin has been isolated, andthe analysis has been completed. The cDNA sequence has been confirmed ashuman lactoferrin cDNA by comparison of the deduced amino acid sequencewith the published amino acid sequence of hLF. The expression oflactoferrin was observed in a eucaryotic expression system from the cDNAand a plasmid vector. The presence of lactoferrin was confirmed bystandard Western immunoblot analysis using anti-human lactoferrinantibodies and relative molecular mass measurement.

FIG. 1 is a schematic of the lactoferrin cDNA. The sequence cangenerally be described as an initial 5′ untranslated region, 17nucleotides in length. The next portion is 57 nucleotides which codesfor the 19 amino acid secretion signal peptide starting with methionine.The next sequence of the cDNA codes for the mature human lactoferrinprotein of 692 amino acids followed by the 3′ untranslated region of 208nucleotides which ends the cDNA. The complete sequence is 2,358nucleotides in length. The hLF protein contains glycosylation sites. ThehLF protein with secretion signal sequence has an expected molecularmass of 78,403 daltons and the mature hLF is 76,386 daltons withoutadded carbohydrate from glycosylation.

FIG. 2 is the cDNA sequence (SEQ ID No. 1) with the deduced amino acids(SEQ ID No. 2) for the secretion signal peptide and the mature humanlactoferrin protein. The numbers on FIG. 2 correspond to the nucleotidesstarting at the 5′ end. There are binding sites for two iron atoms withfour amino acids participating in the binding of each iron. The aminoacids at positions Asp80, Tyr112, Tyr209, and His273 are required forcoordination with one iron, and amino acids at positions Asp415, Tyr455,Tyr548, and His6l7 bind the other. There are two glycosylation sites atpositions Asn157 and Asn498. The numbers refer to the deduced amino acidsequence. There are 25 amino acids per line of protein sequence(starting at nucleotide 18).

The nucleotide sequence analysis was performed on cDNA isolated from ahuman prostate cDNA library. The prostate cDNA library yielded a 2,140bp cDNA which contained the complete 5′ end including the untranslatedportion and the signal sequence. The 3′ end including the three aminoacids at the carboxy terminal and the untranslated region were obtainedas a 208 bp cDNA from both a monocyte cDNA library and human prostatecDNA library.

The data in FIG. 2 displays the full-length cDNA sequence of thisinvention. The complete sequence including the 5′ untranslated regionand signal peptide have not been reported. Further, the previouslyreported amino acid sequence varies from the deduced amino acid sequencefor hLF of this invention. The following TABLE 1 is a summary of thedifferences of the amino acid sequence of the present invention andthose reported by Metz-Boutigue et al., Eur. J. Biochem., vol. 145, pp.659-76 (1984). For the purpose of this table, the numbering of the aminoacids will be initiated with methionine at the start of the signalpeptide sequence as amino acid #1. TABLE 1 COMPARISON OF AMINO ACIDSEQUENCES HUMAN LACTOFERRIN Amino Acid Deduced Metz-Boutigue from cDNAof hLF Change Sequence # 30 Thr Substitution Ala # 48 Arg SubstitutionLys # 141 Arg Insertion NONE # 170 Ala Insertion NONE # 204 SerSubstitution Leu # 206 Gln Substitution Lys # 209 Tyr Substitution Lys #386 Glu Substitution Gln # 392 Ser Substitution Trp # 410 AspSubstitution Asn # 411-424 Deletion 13 Amino acids in protein sequencenot in deduced amino acid sequence from cDNA # 532 Gln Substitution Glu# 695 Lys Substitution Arg

FIG. 3 is the expression of human lactoferrin protein from the completehLF cDNA. In addition to using the entire cDNA sequence and deducedamino acid sequence, a polypeptide of less than the entire protein canbe of value. For instance, the region between amino acids 74-275contains an iron binding domain which may be used without the rest ofthe protein for biologically available iron or the bacteriostaticqualities.

The cDNA sequence has been confirmed to encode lactoferrin. The hLF cDNAwas shown to encode lactoferrin by expression of the cDNA in aeucaryotic expression system and detection of the expressed lactoferrinprotein by Western immunoblot analysis using specific lactoferrinantibodies.

Recombinant production of lactoferrin protein has been described belowin its preferred embodiments. However, it is also produced in number ofother sources such as fungal sources such as Saccharomyces cerevisiae,Kluyveromyces lactis, or Pichia pastorsis, or insect cells such as SF9,or bacterial cells such as Escherichia coli, or Bacillus, subtilis.

In one embodiment of the present invention, biologically activerecombinant lactoferrin protein is produced. This method comprisessynthesizing sequences containing a selectable marker gene, a promotor,a transcription termination sequence and a linker sequence.

Subsequently, the sequences are cloned to form a plasmid and the plasmidis digested with a restriction endonuclease. A cDNA coding forlactoferrin is inserted into a restriction site and eucaryotic cells arethen transformed with the plasmid expressing the lactoferrin cDNA.

The selectable marker gene useful in the method of the present inventionmay be any that permits isolation of cells transformed with alactoferrin cDNA plasmid. Preferably, the selectable marker gene isselected from pyr4, pyrG, argB, trpC and andS.

The promotor useful in the present invention may be any that allowsregulation of the transcription of the lactoferrin cDNA. Preferably, thepromotor is selected from the group of alcohol dehydrogenase, argB,α-amylase and glucoamylase genes.

The transcription termination sequence useful in the present method maybe any that allows stabilization of the lactoferrin mRNA. Preferably,the transcription termination sequence is derived from the α-amylase,glucoamylase, alcohol dehydrogenase or benA genes.

The linker sequence useful in the present method may be any thatcontains a translation initiation codon, a secretory signal and arestriction enzyme cleavage site. Preferably, the linker element isderived from the α-amylase or glucoamylase genes.

The cells, preferably eucaryotic cells, useful in the present inventionare any that allow for integration of a vector, preferably a plasmidcomprising the lactoferrin cDNA and expression of the lactoferrin cDNA.Preferably, the eucaryotic cells are fungal cells or insect cells.Insect cells such as SF9 are useful in the method of the presentinvention. More preferably, the fungal cells are yeast cells orAspergillus. Most preferably, the eucaryotic cells useful in the presentinvention are Aspergillus strains, such as A. oryzae, A. niger, A.nidulans and A. awamori.

The invention also comprises partial sequences of the cDNA of SEQ ID No.1, 3 and 5 and substitution analogs thereof which code for biologicallyactive polypeptides having homology with a portion of lactoferrin,especially those that are not available from enzyme digests of naturallactoferrins, the method of making polypeptides by use and expression ofpartial cDNA sequences, and the polypeptide products produced by themethods of this invention. The desired partial sequences can be producedby restriction enzyme cleavage, as for example at the cleavage sitesindicated in FIGS. 18, 19 and 20. the partial sequences may also besynthesized or obtained by a combination of cleavage, ligation andsynthesis, or by other methods known to those skilled in the art.

Recombinant production of lactoferrin protein and polypeptides has beendescribed in its preferred embodiment. However, it is also, produced ina number of other sources such as fungal sources such as Saccharomycescerevisiae, Kluyveromyces lactis, or Pichia pastorsis or insect cellssuch as SF9, and lactoferrin polypeptides may also be produced inbacterial cells such as Escherichia coli, or Bacillus subtilis.

The following examples are given for the purposes of illustratingvarious embodiments of the present invention and are not meant to belimitations of the present invention in any form.

EXAMPLE 1 Human Lactoferrin cDNA

The complete 2,358 bp hLF cDNA was ligated to the eucaryotic expressionvector, p91023(B) at the EcoRI site downstream from the adenovirus majorlate promoter. This plasmid vector was provided by Genetics Institute(Cambridge, Mass.) and has been described in previous publications (Wonget al., Science 288:810-815 (1985)). The hLF cDNA expression vector wastransferred into COSM-6 monkey kidney cells using standard tissueculture transfection conditions (Wigler et al., Cell, 16:777-785(1979)). These COS cells do not normally express lactoferrin.Forty-eight hours after transfection, the cells were harvested and crudecell extracts were prepared. Positive identification of the humanlactoferrin was made by standard Western immunoblot analysis of theproteins expressed in the cell extracts, as well as those secreted intothe cell growth medium using a commercially available antibody directedagainst human lactoferrin (Sigma). Proteins which bound to theanti-lactoferrin antibody were detected using radio-iodine labelledProtein A which reacts with the antibody. The immunoblots wereautoradiographed to identify the human lactoferrin protein. FIG. 3 is anautoradiographic film showing the human lactoferrin expressed in fourcell extracts prepared from tissue culture cells which were transfectedwith the lactoferrin cDNA expression vector (lanes 5 to 8). Lanes 5 to 8show that the transfected cells all contain human lactoferrin (markedwith an arrow) which is immunoreactive with the anti-lactoferrinantibody and is the same molecular weight as human lactoferrin(M_(r)=78,403 daltons). The control cells which were not transfectedwith the cDNA did not contain lactoferrin (lanes 3 and 4). Analysis ofthe growth medium showed that human lactoferrin was also secreted intothe medium from transfected cells (lane 2) but not from control cells(lane 1).

The cDNA encodes a recombinant human lactoferrin protein which issimilar to human lactoferrin protein isolated from milk as determined bymolecular size comparisons and immunoreactivity with anti-humanlactoferrin. Furthermore, the secretion signal peptide sequence isfunctional since the human lactoferrin is secreted into the growthmedium of tissue culture cells which express the cDNA.

FIG. 4 is a schematic representation of the human lactoferrin proteinprecipitated after in vitro transcription and translation of the humanlactoferrin cDNA. The 2140 bp cDNA was from the human prostate cDNAlibrary and included the 5′ untranslated region and the rest of the basepairs correlative to the cDNA sequence of FIG. 2 omitting the last 208bp at the 3′ terminus. The 2140 bp cDNA was ligated to the EcoRI site ofthe plasmid vector pGEM₄ (commercially available from Promega Biotech.,Madison, Wis. 53711-5305) downstream from the SP₆ promoter. The plasmidconstruct was linearized at the 3′ end of the hLF cDNA using therestriction enzyme Hinc II or Xba I. The linear DNA template was thentranscribed in vitro using purified SP₆ RNA polymerase in the presenceof ribonucleotides as described in the manufacturers protocol (PromegaCorporation 1988/1989 Catalogue and Applications Guide). The resultantmRNA was translated using 100 ng mRNA template and micrococcal nucleasetreated rabbit reticulocyte lysate (as described by Promega) in thepresence of 75 uCi ³⁵S methionine (800 ci/mmol, Amersham). In vitrosynthesized lactoferrin was immunoprecipitated by incubating 100 ulaliquots of translation reaction with 10 ug of rabbit anti-humanlactoferrin IgG (Sigma Chemical Company, St. Louis, Mo. 63178) for 2hours at 4° C. in 50 mM Tris, pH7.5/0.15M NaCl/0.05% Tween-20 (1Pbuffer). The reaction volume was 200 ul. Immunoreactive lactoferrin wasprecipitated after incubation for 1 hour with 50 ug of Protein Asepharose (Pharmacia, Upsalla, Sweden). Immunoprecipitation was carriedout by centrifugation for 5 minutes at 10,000 g and the precipitate waswashed 5 times with 4 volumes of 1P buffer. Total translation productsand immunoprecipitates were then subjected to electrophoresis indenaturing 7.5% polyacrylamide gels. After fixing in 50% methanol, thegels were incubated in En³Hance (NEN, DuPont, Wilmington, Del. 19801)for 1 hour and washed with distilled H₂O. The gel was then dried undervacuum and exposed to Kodak X-OMAT XAR film at −70° C.

Lane 1 shows ¹⁴C protein molecular weight markers used to estimate thesize of the translated proteins. Lane 2 is a negative control whichshows that no ³⁵S labelled proteins are translated in this system whenno mRNA is added to the translation mix. Lanes 3 and 4 show the totaltranslation products obtained when lactoferrin MRNA is added afterpreparation from two separate DNA templates. The major protein band(marked with an arrow) is human lactoferrin. This is the only banddetected when the translation products are immunoprecipitated withanti-human lactoferrin before applying the protein to the gel (lane 6).The measurement of molecular mass by SDS-PAGE does not correspond toexact molecular weight due to secondary protein structure. However, thevalues are shifted in a correlative manner in comparison to the control.Analysis of the size of the translated lactoferrin is shown in FIG. 4.The protein migrated at the expected molecular mass of human lactoferrin(about 78 Kd). The major bands in lanes 3 and 4 which migrate higherthan the 68 Kd marker band in the control lane correspond to expectedmolecular mass of hLF protein on SDS-PAGE.

EXAMPLE 2 Fungal Strains and Transformation

The pyrG mutant strain used in these studies was derived from A. oryzae(A07 11488). The pyrG gene from A. oryzae was mutated with4-nitroquinoline-1-oxide. The Aspergillus transformation was carried outby, a modification of the procedure of Osmani, et al., J. Cell. Biol.104:1495-1504 (1987). Conidia (1X10⁶/ml) were inoculated into 50 ml ofYG medium (0.5% yeast extract 2% glucose) containing 5 mM uracil and 10mM uridine. Growth was at 32° C. for 14-16 hours until a germ tube wasvisible. The germinated conidia were harvested by centrifugation andresuspended in 40 ml of lytic mix containing 0.4 M ammonium sulphate, 50mM potassium citrate (pH 6.0), 0.5% yeast extract, 0.12 g novozyme, 0.1g Driselase, 100 μl β-glucuronidase, 0.5% sucrose and 10 mM MgSO₄.Protoplasting was for 2-3 hours at 32° C. and 150 rpm. Followingprotoplasting, filtration using sterile miracloth was necessary toremove any undigested mycelia. The protoplasts were harvested bycentrifugation and washed twice with 10 ml of 0.4 M ammonium sulphate,1% sucrose and 50 mM potassium citrate (pH 6.0) at 4° C., resuspended in1 ml of 0.6 M KCl; 50 mM CaCl; 10 mM Tris-HCl (pH 7.5) and placed onice. The transformation was performed immediately following theprotoplast preparation. Aliquots (100 μl) of the protoplast were addedto 3 μg of DNA and 50 μl of 40% polyethylene glycol (PEG) 6000, 50 mMCaCl₂, 0.6 M KCl and 10 mM Tris-HCl, (pH 7.5). The samples wereincubated on ice for fifteen minutes after which an additional 1 ml ofthe PEG solution was added and incubation at room temperature wascontinued for thirty minutes. Aliquots of this mixture were plated in 3mls of 0.7% minimal media, supplemented with 0.4% ammonium sulphate ontoplates containing the same but solidified with 2% agar. All subsequentgrowth was at 32° C.

EXAMPLE 3 Plasmid Construction

A schematic representation of the expression plasmid is shown in FIG. 5.The complete cDNA encoding human LF was repaired using the Klenowfragment of DNA polymerase I and subcloned into Acc I digested andrepaired pGEM4 to generate pGEMhLFc. In order to remove the LF signalsequence and generate a 5′ end in frame with the a-amylase sequences, a252 base pair lactoferrin fragment (nt 69-321) containing Hind II/Acc Iends was obtained by polymerase chain reaction (PCR) amplification ofpGEMhLFc plasmid DNA. The oligo primers used were as follows: the 5′ endoligonucleotide as shown in SEQ. ID. No. 7:(CTGGGTCGACGTAGGAGAAGGAGTGTTCAGTGGTGC)

and the 3′ end oligonucleotide as shown in SEQ. ID. No. 8:(GCCGTAGACTTCCGCCGCTACAGG).

This PCR fragment was digested with Hind II and Acc I and was subclonedinto Hind II/Acc I digested pGEMhLFc generating pGEMhLF. A 681 base pairα-amylase fragment with Asp718/Pvu II ends encoding the promotor, signalsequence and the alanine residue from the start of the mature α-amylaseII gene, was obtained by PCR amplification of A. oryzae genomic DNA. Theoligo primers were as follows: the 5′ end oligonucleotide as shown inSEQ. ID. No. 9: (GAGGTACCGAATTCATGGTGTTTTGATCATTTTAAATTTTTATAT)

and the 3′ end oligonucleotide as shown in SEQ. ID. No. 10:(AGCAGCTGCAGCCAAAGCAGGTGCCGCGACCTGAAGGCCGTAC).The amplified DNA was digested with Asp718 and Pvu II and subcloned intoAsp718/Hind II digested pGEMhLF. The resulting plasmid (pGEMAhLF) wasdigested with EcoR I and the resulting 2.8 kb α-amylase-lactoferrinfragment was subcloned into a unique EcoR I site in pAL3 according tothe method of generating pAhLF*. Synthetic oligonucleotides were used toprovide the last five carboxy terminal codons of lactoferrin (nt2138-2153) missing in pAhLF* and also to provide the first 180 bp of 3′untranslated sequences from the A. niger glucoamylase gene. Theresulting plasmid (pAhLFG) was used to transform the A. oryzae pyrGmutant strain.

With reference to FIG. 5, Aspergillus oryzae expression plasmid, pAhLFGcontains 681 bp of 5′-flanking sequence of the A. oryzae AMY II genewhich includes the signal sequence and first codon of mature α-amylase.The cDNA coding for mature human lactoferrin is subcloned in framedownstream from these sequences allowing recombinant protein productionby the addition of starch to the growth medium. The Aspergillus nigerglucoamylase 3′ untranslated region provides the transcriptionterminator and polyadenylation signals. The plasmid also contains theNeurospora crassa pyr4 selectable marker and an ampicillin resistancegene.

The plasmid construct (pAhLFG) used for expression of human LF containsa 681 bp fragment that encodes the promotor and secretory signal peptideof the A. oryzae α-amylase II gene (AMY II). The signal sequence alsocontains the codon for alanine from the start of the ct-amylase matureprotein generating the signal sequence cleavage site (Leu Ala)recognizable by an endogenase α-amylase peptidase. A human lactoferrincDNA fragment encoding the mature protein was subcloned in frameimmediately downstream from the AMY II sequences, placing it under thecontrol of this highly efficient starch inducible promoter. In order tostabilize the transcribed human LF mRNA, a 180 bp fragment encoding the3′ untranslated region of the glucoamylase gene from Aspergillus nigerwas ligated into a unique BamH I site in the multiple cloning cassette,immediately downstream of the human LF cDNA providing the transcriptionterminator and polyadenylation signals. The plasmid also contains theNeurospora crassa pyr4 selectable marker which complements a pyrgauxotrophic mutation of A. oryzae and allows for selection of sporesthat have been transformed with the plasmid by growth in the absence ofuridine.

EXAMPLE 4 Genomic DNA Manipulation

A. oryzae DNA was isolated from 200 mg of lyophilized mycelia asdescribed by Rasmussen, et al., J. Biol. Chem., 265:13767-13775 (1990).The DNA was digested with EcoR I, size fractionated on a 0.8% agarosegel and transferred to nitrocellulose. Prehybridization andhybridization of the nitrocellulose filter for Southern analysis wereperformed in 6×SSC, 0.1% SDS and 0.5% dried milk at 65° C. for 16 hours.Hybridization solution contained 1×10⁷ cpm ³²P-labelled lactoferrin cDNAprobe (2.1 Kb). The filter was washed in 2×SSC, 0.5% SDS at roomtemperature for 30 minutes followed by two washes in 0.5×SSC, 0.5% SDSat 68° C. for 30 minutes. The filter was dried, exposed at −70° C. fortwo hours and developed by autoradiography.

With reference to FIG. 6, Southern blot analysis was performed ontransformed Aspergillus oryzae strains. Genomic DNA from individualtransformants and control AO7 were hybridized with a radiolabelled hLFcDNA probe (2.1 kb). The arrow points to a radiolabelled fragment (2.8kb) generated upon EcoR I digestion of the expression plasmid which ispresent in all the transformants (#1-9) but is absent in controluntransformed AO7. Molecular weights of bacteriophage lambda Hind IIIfragments are indicated at the left.

EXAMPLE 6 Northern Analysis

RNA was isolated from lyophilized mycelia (200 mg) using commerciallyavailable RNazol B (Biotecx Laboratories, INC, Houston, Tex.) accordingto the manufacturers instructions. Total RNA (20 μg) was electrophoresedin a 0.8% agarose gel containing 2.2 M formaldehyde. The RNA wastransferred to nitrocellulose and hybridized with either a 2.1 kblactoferrin cDNA or a 1.8 kb genomic α-amylase fragment corresponding tothe coding region of the α-amylase II gene. The probes were ³²P-labelledby nick translation (specific activity 2×10⁸ cpm/ug). Hybridization wascarried out 2×SSC, 0.05% dried milk at 65° C. over an ice with 2×10⁶ cpmprobe/ml.

Washes were identical to those employed in the Southern analysis. Thefilters were dried, exposed at −70° C. for two hours and developed byautoradiography. RNA dot blots were performed using nitrocellulosemembrane and the manifold dot blot system. Hybridization and washingconditions were as described above for Southern analysis. Radioactivitywas quantitated using the betagon blot analyzer.

With reference to FIG. 7, RNA analysis of transformant versus controlAO7 was performed. In Panel A, Northern analysis of RNA (20 μg) fromcontrol AO7 and transformant #1 were hybridized with radiolabelled humanLF cDNA. Human LF mRNA (2.3 kb) was detected in the transformant #1 butnot in the control untransformed AO7. The positions of the 28S and 18SrRNA bands are indicated on the left. In Panel B, Dot blots of RNA (5and 10 μg) from control AO7 versus transformant #1 using a radiolabelledα-amylase genomic DNA probe. In Panel C, Dot blots of RNA (5 and 10 μg)from control A07 and transformant #1 using radiolabelled human LF cDNAprobe as illustrated.

Northern analysis was performed to determine if lactoferrin mRNA wastranscribed correctly and efficiently in A. oryzae under the regulatorycontrol elements of the expression plasmid. Spores (1×10⁶/ml) fromtransformant #1 and from control untransformed spores were inoculatedinto fungal medium containing 1.5% glucose. as carbon source and grownat 30° C. for 48 hours in small shake flask cultures. The cultures werewashed and reinoculated into fungal medium containing 3% starch toinduce transcription of the human LF MnRNA. After 24 hours, the cellswere harvested and RNA was isolated. Total RNA (20 μg) was sizefractionated on a 1.0% agarose gel containing 2.2 M formaldehyde andblotted on nitrocellulose.

Human lactoferrin mRNA was detected using ³²p labelled human LF cDNA(2.0 kb) probe. Hybridization with human LF radiolabelled cDNA probedetected a specific radiolabelled band at the correct size forlactoferrin MRNA (2.3 kb) in the transformant but not in the controluntransformed strain (FIG. 7A). Quantitation of mRNA levels by dot assayshowed comparable levels of expression of endogenous α-amylase rRNAbetween control AO7 and transformant #1 (FIG. 7B). In addition, similarlevels of expression of α-amylase and human LF mRNA were seen intransformant #1 (FIG. 7B and 7C).

EXAMPLE 6 Purification of Recombinant Human LF

LF was purified from the growth medium using CM Sephadex C50 essentiallyas described by Stowell, et al., Biochem J., 276:349-59 (1991). Thecolumn was pre-equilibrated with 500 ml of 0.025 M Tris HCl, pH 7.50 1MNaCl. The pH of the culture medium was adjusted to pH 7.4 beforeapplying to the pre-equilibrated column. The column was washed with 500ml of equilibration buffer and followed by a linear salt gradient from0.1 to 1.1 M NaCl. Fractions (7 ml total) were assayed for lactoferrincontent and purity using SDS/PAGE and silver staining. Fractionscontaining LF were dialyzed against 0.025 M Tris HCl, pH 7.5/0.1M NaCland lyophilized.

EXAMPLE 7 Quantitation of Human LF

Recombinant lactoferrin was quantitated using an ELUSA assay essentiallyas described by Vilja et al., J. Immunol. Methods, 76:73-83 (1985). Asensitivity of 5 ng of lactoferrin was obtained using thenon-competitive Avidin-biotin assay. Human LF isolated from breast milk(Sigma) was used as standard. Biotinylated human lactoferrin IgG wasobtained from Jackson Immunoresearch laboratories, West Grove, Pa.

EXAMPLE 8 N-Terminal Sequencing

Five μg of purified recombinant human LF was resolved on anSDS-polyacrylamide gel and transferred to Problott, a polyvinylidenedifluride-type membrane, following manufacturers instructions (AppliedBiosystems). Human LF was detected with Comassie Brilliant Blue stainingand destained. This human LF band was excised, washed thoroughly withdistilled H₂O and air-dried. The N-terminal amino acid sequence of thefirst ten amino acids of human LF was determined by the automated Edmandegradation procedure using an applied Biosystems Pulsed-liquid phasesequencer (Model 477A).

With reference to FIG. 8, panel A illustrates a Silver stainedSDS-polyacrylamide gel analysis of recombinant human LF secretion andpurification. Lane 1 contains breast milk human LF standard (500 ng).Lanes 2 and 3 contain samples of the growth medium (40 μg) from inducedcontrol AO7 and transformant #1 respectively. Lanes 4-8 contain 100 μlaliquots of eluted fractions (#25, 30, 35, 40, and 45 respectively)collected from the CM-sephadex purification of recombinant LF from thegrowth medium of transformant #1. The position of the molecular weightmarkers (BioRad Richmond, Calif.) are indicated on the left. Sizes aregiven in kilo Daltons. Panel B illustrates a Western immunoblot analysisof duplicate samples as described in panel A using a specific polyclonalantibody directed against human LF with detection with ¹²⁵I-protein A.Panel C illustrates #6 N-terminal amino acid sequence of recombinanthuman LF. Recombinant human LF was sequenced from the N-terminus through10 residues and is identical to breast milk human LF with the exceptionof the additional alanine generated in our construction to provide theα-amylase signal sequence cleavage site.

EXAMPLE 9 Deglycosylation

Deglycosylation was performed using N-glycosidase F (BoehringerMannheim). A. oiyzae growth medium containing 0.5 μg lactoferrin wasdenatured for 3 minutes at 100° C. in the presence of 0.01 % SDS.Standard LF from human milk was treated similarly. The samples weresubsequently placed on ice for five minutes. N-glycosidase F reactionswere conducted in 0.4 M sodium phosphate, (pH 6.8); 0.08% Triton; 0.1%β-mercaptoethanol and 1 unit of enzyme and incubated at 37° C. forsixteen hours. PAGE and Western analysis was performed using an IgGspecifically directed against human lactoferrin to detect an increase inmobility of digested samples.

With reference to FIG. 9, recombinant human LF was characterized. PanelA illustrates the deglycosylation of lactoferrin. Western analysis ofglycosylated and deglycosylated lactoferrin using a specific polyclonalantibody was directed against human lactoferrin with detection with¹²⁵I-protein A. The first panel contains authentic breast milk human LF(500 ng) untreated (−) and treated (+) with N-glycosidase F. The secondpanel contains purified recombinant human LF (500 ng) untreated (−) andtreated (+) with N-glycosidase F. The size of glycosylated human LF isindicated with the arrow. Panel B illustrates a functional analysis ofrecombinant lactoferrin with regard to iron-binding capacity. Panel Aand B show the ⁵⁹Fe filter binding assay of duplicate samples ofauthentic breast milk human LF and purified recombinant human LF,respectively, at the concentrations indicated. The first lane in bothpanels contain BSA (5 μg) as a negative control.

Lactoferrin contains two N-acetyllactamine type glycans attached throughN-glycosidic linkages. To determine if recombinant lactoferrin wasglycosylated correctly, the protein was treated with N-glycosidase F,resolved on SDS-polyacrylamide electrophoresis, transferred tonitrocellulose and probed using a specific IgG directed against humanlactoferrin (FIG. 11A). N-glycosidase F hydrolyses at the glycosylaminelinkage generating a carbohydrate free peptide of smaller molecularweight. Comparison of recombinant LF with purified LF from human milk,illustrates that both proteins co-migrate upon digestion withN-glycosidase F suggesting that the recombinant protein has aglycosylation pattern similar to native LF.

Lactoferrin has a bilobal structure with each lobe having the capacityto bind tightly, but reversibly, one Fe³⁺ ion. The iron-bindingproperties of lactoferrin are crucial for its functional roles. To testif recombinant human LF expressed and secreted in A. oryzae has an ironbinding capacity similar to authentic lactoferrin, an ⁵⁹Fe micro filterbinding assay was developed. Purified human lactoferrin isolated fromthe growth medium of transformant #1 was dialyzed against 0.1M citricacid (pH 2.0) to generate apo-human LF. Native lactoferrin from humanmilk was treated similarly. Excess ⁵⁹Fe (0.2 mCi) was added to thesesamples in an equal volume of 1 M bicarbonate, followed by incubation at37° C. for 30 minutes. Samples were applied to nitrocellulose membraneand washed several times with bicarbonate. The filter was visualized byautoradiography and Fe-binding was quantitated using a betagon blotanalyzer. As illustrated in FIG. 11B, both recombinant and native LFshowed a similar level of iron binding at all concentrations tested. Theresults demonstrate that recombinant human LF is indistinguishable fromnative human LF in its capacity to bind iron.

With reference to FIG. 2, the complete cDNA sequence for humanlactoferrin protein is depicted. The cDNA coding for lactoferrin is usedto create plasmids and transform eucaryotic cells and to produce thelactoferrin protein.

Strains of Aspergillus used in the present invention are auxotrophicmutants that contain a defective pry4 gene that results in an inabilityto synthesis orotidine 5′ phosphate (OMP) decarboxylase. The enzyme isrequired for uridine synthesis. The strain cannot grow on media lackinguridine. The plasmid contains a selectable marker, i.e., a sequence thatencodes the gene for OMP decarboxylase. Uptake of the plasmid by theAspergillus can therefore be selected for by growth on media lackinguridine. The Aspergillus is transformed by the plasmid such that it cangrow on the uridine deficient media.

EXAMPLE 10 Expression of the 3′ Iron-Binding Domain of HumanLactoferrin-E. Coli

The 3′ iron-binding domain of human lactoferrin (hLF) was expressed inEscherichia coli using the bacterial expression plasmid, PT7-7 asdescribed by Tabor, S. and Richardson, C., Proc. Natl. Acad. Sci.U.S.A., 82:1074-1078 (1985). pGEMhLFc, containing the cDNA for thecomplete hLF cDNA (Ward, P. P., et al. Gene. 122:219-223 (1992)), wasdigested with Sma I and Hind III to release a 1.5 kb fragment encodingthe 3′ iron-binding domain of hLF. This 1.5 kb Sma I/Hind III fragmentwas subcloned in-frame into Sma I/Hind II digested PT7-7, under thecontrol of the strong inducible T7 promoter, generating PT7-7hLF3′.

PT7-7hLF3′ was transformed into a protease deficient strain of E.coliwhich had previously been transformed with pGP1-2 plasmid whichcontained the T7 polymerase under the control of the λpL promoter asdescribed by Conneely, O. M., et al. In: Hormone Action and MolecularEndocrinology. 5-48-5-50 (1989)). The PT7-7 plasmid contained anampicillin resistance gene while the pGP1-2 plasmid contained akanamycin resistant gene allowing dual antibiotic resistance selectionfor transformants containing both plasmids. Transformants obtained werecultured overnight in LB broth containing ampicillin (50 μg/ml) andkanamycin (50 μg/ml) at 30° C./250 rpm. Overnight cultures weresubcultured into LB (500 ml) containing ampicillin and kanamycin andgrown at 30° C./250 rpm until an O.D.₆₀₀nm of 0.5-0.6 was obtained. At30° C. the λ repressor bound to the λpL promoter, thus blocking T7polymerase production. Induction of the recombinant protein was achievedby raising the temperature to 42° C. for one hour to inactivate the λrepressor thus allowing T7 polymerase production. The temperature waslowered to 30° C. for a further two hours, turning off λpL directedtranscription and allowing the production of the recombinant protein asthe T7 polymerase bound to the T7 promoter to specifically induceexpression of the recombinant lactoferrin 3′ iron-binding domain.

Western Immunoblot analysis was performed to determine if the 3′ ironbinding domain was expressed in the bacterial cells under the control ofthe T7 promoter and to monitor its purification. The cells wereharvested at 5000 g and resuspended in 15 ml of PBS (pH 7.4). Totalcellular extracts were prepared by sonication for 1 minute on ice. Thesonicate was centrifuged at 13,000 g for 40 minutes at 4° C. Thesupernatant was removed and the pellet was resuspended in 50 ml ofdenaturation buffer (5M urea, 2% triton, 5 mM EDTA, 0.01% Tween 20, 50mM TrisCl, pH 7.5) and centrifuged at 48,000 g for one hour. Thesupernatant containing the soluble fraction was recovered. Proteinconcentration was determined using the Bradford reagent according tomanufacturers instructions (BioRad, Richmond, Calif.). Protein samples(40 μg) were resolved by SDS-PAGE and transferred to a nitrocellulosefilter electrophorectically using the Western Immunoblot procedure. Thefilter was blocked with Tris-buffered saline (TBS, 0.05 M Tris/0.15 MNaCl, pH 7.5) containing 2% dried milk, and then incubated for 2 hoursin the same with the addition of a specific polyclonal IgG (1 μg/ml)directed against hLF (Sigma, St. Louis, Mo.). The filter was washed(5×10 min) in TBS/0.05% Nonidet P40 followed by incubation with 5 μCi of¹²⁵I protein A in TBS/2% dried milk. The filter was washed (5×10 min) inTBS/0.05 % Nonidet P40, dried and exposed overnight in Kodak XAR5 filmat −70° C. The film was developed by autoradiography.

The results of the Western analysis are shown in FIG. 10. Animmunoreactive band at the expected size (50 kDa) for the hLF 3′iron-binding domain was evident in the cellular extract from inducedcells and was absent in control uninduced cells (FIG. 10, lanes 1 and2). The hLF 3′ iron-binding domain associates with the cellularhomogenate insoluble fraction (FIG. 10, lane 3) and hence required afurther solubilization step in a denaturation buffer to prepare the hLFin a soluble form (FIG. 10, lane 4).

Analysis of a coomassie-stained SDS-PAGE gel also showed the presence ofa 50 kDa protein in the cellular extract from induced cultures which wasabsent in control uninduced cultures (FIG. 11, lanes 2 and 3). Therecombinant protein was expressed at levels up to 10 mg/l andrepresented approximately 5% of the total cellular protein. The hLF 3′iron-binding domain did not associate with the soluble homogenatefraction (FIG. 11, lane 4) and hence required a further solubilizationstep in a denaturation buffer to prepare the hLF in a soluble form (FIG.11, lane 5). Purification and solubilization of the recombinant hLF 3′iron-binding domain resulted in a 50% yield of recoverable protein andrepresented the major protein band in this fraction.

In summary, we have successfully produced recombinant hLF 3′iron-binding domain in E.coli under the control of the strong inducibleT7 promoter. The recombinant protein was expressed and purified in asoluble form from the cellular extracts at levels up to 5 mg/l.

EXAMPLE 12 Expression and Purification of an N-Terminal LactoferrinFragment (AA 1-52) in Escherichia Coli

An N-terminal human lactoferrin fragment (AA 1-52), encoding thebactericidal domain of hLF, reported by Bellamy et al., supra, wasexpressed and purified from E. coli. The bovine lactoferrin fragmentalso reported by Bellamy, et al. is produced by the same methodillustrated here for the human fragment. This was achieved using theglutathione S-transferase (GST) Gene Fusion System (Pharmacia,Piscataway, N.J.) where the lactoferrin fragment was expressed as afusion protein with glutathione S-transferase [Smith, D. S., et al.,Gene, 67:31-40 (1988)] and a protease cleavage site allowing productionof the bactoricidal domain by cleavage from GST.

A 156 bp human lactoferrin fragment encoding AA 1-52, containing SmaI/BamH I ends was obtained by polymerase chain reaction (PCR)amplification of pGEMhLFc plasmid DNA [Ward, P. P., et al.,Biotechnology, 10:784-789 (1992)]. The oligonucleotide primers used wereas follows: 5′ end oligonucleotide as shown in SEQ. ID. NO. 11CTGCCCGGGCGTAGGAGAAGGAGTGTT 3′ end oligonucleotide as shown in SEQ. ID.No. 12 CATGGATCCTGTTTTACGCAATGGCCTGGATACA

This PCR fragment was digested with Sma I and BamH I and repaired usingthe Klenow Fragment of DNA polymerase I. This fragment was subclonedinto BamH I repaired pGEX-3X generating pGEX-3XLFN-1. This fused thelactoferrin cDNA fragment in frame, downstream from the glutathioneS-transferase gene and under the control of the strong, inducible tacpromoter. All PCR amplified products and construction junctions weresequenced using the commercially available Sequenase version 2.0 kit(United states Biochemical Corp, Cleveland, Ohio).

pGEX-3XLFN-1 was transformed into the bacterial strain, JM109.Transformants obtained were cultured overnight in LB (50 ml) containingampicillin (50g/ml) at 37° C./250 rpm. Overnight cultures weresubcultured into LB (500 ml) containing ampicillin (50 g/ml) and grownat 37° C./250 rpm until an 0D₆₀₀nm of 0.6-0.8 was obtained.Isopropyl-D-thiogalactopyranoside (IPTG) was added to the culture mediumat a concentration of 1 mM to turn on the tac promoter resulting inexpression of the glutathione S-transferase/LFN-1 fusion protein. Growthunder these conditions continued for 4 hours after which the cells wereharvested at 5,000 g and resuspended in 5 ml of MTPBS (150 mM NaCl, 16mM Na₂HPO₄, 4 mM NaH₂PO₄, 1% Triton X-100, pH 7.3). Total cellularextracts were prepared by 3×1 minute freeze/thaw cycles followed by mildsonication for 2×1 minute. The sonicate was centrifuged at 13,000 g for20 minutes and the supernatant obtained was applied to a glutathionesepharose 4B column following manufacturer's instructions (Pharmacia,Piscataway, N.J.). The glutathione S-transferase/LFN-1 fusion proteinwas eluted from the column using 10 ml of elution buffer (10 mMglutathione, 50 mM Tris pH 8.0). Fractions of 1.5 ml were collected anddialyzed overnight against 50 mM Tris, 15% glycerol pH 8.0.

Samples from the solubilized extracts and the purification fractionswere analyzed by SDS/PAGE followed by silver-staining. The results ofthis analysis are shown in FIG. 12. A band at the expected size (32 kDa)for the glutathione S-transferase/LFN-1 fusion protein was detected inthe solubilized protein extracts from induced JM109 cultures transformedwith pGEX-3X/LFN-1 and was absent in uninduced cultures (FIG. 12A, lanes2 and 3). This band migrates at a higher mobility than control inducedJM109 cultures transformed with pGEX-3X alone (FIG. 12A, lane 1). Thefusion protein was successfully purified to homogeneity over aglutathione sepharose 4B column (FIG. 12B, lanes 1 and 2). Proteinconcentration determination using the Bradford reagent (BioRad,Richmond, Calif.) showed that the glutathione S-transferase/LFNI fusionprotein was purified at levels up to 5 mg/l. The GST fusion protein hasa protease cleavage site for the protease Kex II between GST and the 52amino acid protein.

In summary, a human lactoferrin fragment, encoding a bactericidal domainof this protein, has been successfully expressed as a fusion proteinwith glutathione S-transferase an E. coli expression system. This fusionprotein was purified to homogeneity at levels up to 5 mg/l. Thebactericidal protein is obtained by cleavage with the protease Kex II tocleave the GST portion from the bactericidal domain.

EXAMPLE 13 Expression of Bovine and Porcine Lactoferrin in AspergillusOryzae

A universal A. Oryzae expression vector is constructed to allow in framesubcloning of any cloned cDNA of interest. This vector, pAG, is similarto the vector pAhLFG(+1) utilized for the expression of humanlactoferrin in A. Oryzae above. A 680 bp α-amylase fragment encoding thepromoter, signal sequence and the alanine residue from the start of themature -amylase II gene, is obtained by polymerase chain reaction (PCR)amplification of pAhLFG(+1). The oligonucleotide primers are as follows:5′ end oligonucleotide, SEQ. ID. NO. 13 5° CGGAATTCATGGTGTTTGATCATIT 3′end oligonucleotide, SEQ. ID. NO. 145′TGGAATTCGATCGCGGATCCGCAATGCATGCAGCCAAAGCAGGTGCCG CGAC

The 5′ end oligonucleotide encodes an EcoR I site and the 3′ endoligonucleotide contains an Nsi I site, flanked by a BamH I site. Thisamplified DNA is digested with EcoR I and BamH I and subcloned into EcoRI/BamH I digested pAhLFG(+1) generating pAG. All PCR amplified productsand construction junctions are sequenced using the commerciallyavailable Sequenase version 2.0 kit (United States Biochemical Corp.,Cleveland, Ohio).

A schematic representation of this expression plasmid is outlined inFIG. 13. Restriction enzyme digestion of this expression plasmid withNsi I, followed by repair using DNA polymerase I allows subcloning ofany cDNA of interest in frame with the α-amylase signal sequence andalanine residue from the start of the mature α-amylase II gene. 5′ and3′ oligonucleotide primers are designed to contain Acc 1 ends, and usedto obtain the full length cDNA encoding for mature porcine and bovinelactoferrin using polymerase chain reaction (PCR) amplification of theirknown DNA sequence. The PCR fragment thus obtained is digested with AccI and repaired using the Klenow fragment of DNA polymerase I for inframe subcloning into Nsi I blunt-ended pAG. The plasmids are then betransformed into the pyrG- strain of A. Oryzae to obtain expression andsecretion of these cDNAs as previously described for human lactoferrin.

EXAMPLE 14 Expression of Human Lactoferrin in Saccharomyces Cerevisiae

The complete human lactoferrin (hLF) cDNA was expressed in Saccharomycescerevisiae using the yeast expression plasmid, YEP [McDonnell, D. P. etal., J. Steroid Biochem, Molec. Biol., 39:291-297 (1991)]. A 2.2 kbfragment encoding the complete hLF cDNA SEQ. ID No. 1 was generatedusing the polymerase chain reaction. This fragment contained and XhoIrestriction enzyme site at its 5′ end and an Asp718 restriction enzymesite at its 3′ end. The 2.2 kb fragment was subcloned, in frame, intoXhoI/Asp718 digested YEP to yield, YEPLFc.

Transcription of the hLF cDNA was under the control of the copperresponsive yeast metallothionein promoter (CUP1). hLF was produced as aubiquitin fusion protein. The fusion protein is short lived in the yeastcells and is processed to produce unfused protein upon folding.

YEPLFc was transformed into a protease deficient strain of S.cerevisiae,by standard techniques [Ito, H., et al., J. Bacteriol., 153:163-186(1983).] This strain cannot grow unless the growth medium issupplemented with adenine, uracil and tryptophan. The YEP plasmidcontains a tryptophan selectable marker, thus, transformants wereselected by tryptophan auxotrophy.

Transformants obtained were cultured overnight in selective mediumcontaining 2% glucose, 0.1% casamino acids, 0.67% yeast nitrogen base,0.001% adenine and 0.002% uracil at 30° C./200 rpm. When the cellsreached an 0D₆₀₀nm of 1.0, 1×10⁶ cells were inoculated into 10 ml of theselective medium and 100 μm CuS0₄ added. The cells were grown for 24hours at 30° C./200 rpm. The purpose of adding the CuS0₄ was to induceexpression of the hLF cDNA from the copper inducible CUP1 promoter.

Western immunoblot analysis was performed to determine if hLF wasexpressed in the yeast cells under the control of the CUP1 promoter. Thecells were harvested by centrifugation at 5000×g for 5 min. andresuspended in 1 ml of Z buffer (120 mM Na₂HP0₄7H₂O, 40 mM NaH₂PO₄H₂O,10 mM KCl, 1 mM MgSO₄7H₂O, 0.27% 2-mercaptoehanol, pH 7.0). Totalcellular extracts were prepared by glass bead homogenization. Thisprocedure involved mixing the yeast cells with an equal volume of glassbeads (0.5 mm, B.Braun Instruments) and vortexing for 5×1 min. Thehomogenate was centrifuged at 13,000 g for 10 min. and the supernatantremoved. The protein concentration was determined using the Bradfordreagent in accordance with the manufacturer's instructions (BioRad,Richmond, Calif.). Protein samples (50 μg) were resolved by SDS-PAGE andelectrophoretically transferred, overnight, to a nitrocellulose filterusing the western immunoblot procedure. The filter was blocked withtris-buffered saline (TBS=0.05M Tris/0.15M NaCl, pH 7.5) containing 1%dried milk and then incubated overnight, in the same, with the additionof a specific rabbit polyclonal antibody (1 μg/ml) directed against hLF(Signa, St. Louis, Mo.). The filter was washed in TBS/0.1% Tween 20 (5×5min.) followed by incubation with horseradish peroxidase (Amersham, UK)for 1 hour. The filter was washed in TBS/0.3% Tween 20 (3×5 min.) andthen TBS/0.1% Tween 20 (3×5 min.). The filter was then treated withluminol and enhancer (Amersham, UK) for 1 min., dried and exposed for 1min. to X-ray film. The film was developed by autoradiography.

These data demonstrate successful production of recombinant hLF in S.cerevisiae under the control of the copper inducible (CUP1) promoter.

The results of the western analysis are shown in FIG. 16. Animmunoreactive band at the expected size (78 kDa) for hLF was evident inthe cellular extract from transformed S. Cerevisiae cells. FIG. 16, lane1.

EXAMPLE 15 Expression of hLF in Aspergillis Nidulans

Construction of the Aspergillis Nidulans Expression Plasmid.

The plasmid used for expression of hLF cDNA is shown schematically inFIG. 17. The cDNA of SEQ. ID No. 1 as a 2.3-kb clone contained thesecretory signal sequence and complete translation frame. The sequenceof the entire cDNA was confirmed by dideoxy sequence analysis (Sequenaseversion 2.0, U.S. Biochemical, Cleveland, Ohio). The cDNA was repairedusing the Pollk and subcloned into AccI-digested and blunt-ended pGEM4.The plasmid, pGEMhLF, was digested with HindIII+Asp718 and repairedusing Polk. The resulting 2.3-kb hLF fragment was subcloned into aunique SmaI site located in the multiple cloning cassette of pAL3downstream from the alcA promoter, Waring, R. B., et al., Gene, 79,119-130 (1989), generating pAL3hLF. The β-tubulin transcriptionterminator fragment was obtained by digesting the 3′-untranslated regionof the bena gene (nt 2569-2665; May et al., 1987) with Xbal+NheI andsubcloned into XbaI-digested pAL3hLF generating pAL3hLFT. This plasmidwas used to transform A. nidulans strain GR5 (pyrG89; wa3; pyroA4)

The A.nidulans expression plasmid, pAL3hLFT, contains 300 bp of55′-flanking sequence of the A. nidulans alcA gene containing all theregulatory elements necessary for controlled gene expression. Toconstruct pALhLFT, a 2.3-kb hLF cDNA fragment containing 17 nucleotidesof 5′-UTR, the complete hLF ORF encoding the secretory signal peptideand mature hLF, followed by 209 nt of 3′ UTR was subcloned into a uniqueSmal site in pAL3 downstream from the alcA promoter. A 96-bp terminatorfragment from the A. nidulans β-tubulin-encoding (benA) gene wassubcloned into a unique XbaI site downstream from the hLF cDNA sequence.The plasmid also contains an Ap^(R) maker and the N. crassa pyr4selectable marker (Waring et al., supra, 1989).

Transformation and Southern Analysis

Transformation was carried out as described by May et al., J. CellBeol., 109, 2267-2274 (1989). Protoplasts were transformed with 3 μg ofthe expression plasmid with an efficiency of 40 transformants/μg DNA.Transformats obtained were purified three times through conidial spores.Southern blot analysis was performed to confirm that transformantscontained integrated plasmid with hLF cDNA. A hLF-specific radiolabelledband was detected at the expected size (2.3 kb) in lanes 1-10 but not inDNA from control spores. These results demonstrate that hLF cDNA wasintegrated into the genome of all A. nidulans transformants tested andvaried randomly from one copy (transformants Nos. 3, 6 and 10) to 20copies (No. 5) per cell. The site of integration of the plasmid into theA. nidulans genome is random due to the absence of homologous sequencesto target the vector into a particular site.

Southern blot analysis was conducted of transformed A. nidulans. GenomicDNA was isolated from ten individual A. nidulans (GR5) transformats anduntransformed spores as described by Rasmussen, C. D. et al., J. Biol.Chem., 265, 13767-13775 (1990). The DNA (1 μg) was digested with EcoRI,size fractionated on a 0.8% agarose gel and transferred to anitrocellulose filter and hybridized with a radiolabelled hLF cDNA probe(2.1-kb). A sample (20 ng) of hLF cDNA was used as a positive control(hLF cDNA). Prehybridization and hybridization of the filter wasperformed in 6×SSC/0.1% SDS/0.5% dried milk at 65° C. for 16h. Thehybridization solution contained 200 ng of ³²P probe (2.1 kb; specificactivity 4×10⁸ cpm/μg of DNA). Filters were washed in 2×SSC/0.5% SDS at68° C. for 30 min followed by 0.5×SSC/0.5% SDS at 68° C. for 30 min. Thefilter was dried and exposed to Kodak X-AR5 film at −70° C. for 30 minand developed by autoadiograpy. The autoradiography showed an intense2.1 kb band for hLF.

Production of hLF in Aspergillus Nidulans

Conidia (1×10⁶/ml) were cultured in minimal media utilizing 100 mM Naacetate pH 6.5 as carbon source with or without addition of 1.2% ethanolto induce transcription of the hLF cDNA. GR5 was cultured as aboveexcept for the addition of 5 mM uridine and 10 mM uracil. Media andmycelia were harvested and separated using Miracloth (Calbiochem, SanDiego, Calif). Mycelia (200 mg) were freeze-dried and lyophilizedovernight. Total cellular extracts were prepared by homogenization in aglass teflon homogenizer using 1 ml of phosphate-buffered saline (PBS;137 mM NaCl/2.7 mM KCl/4.3 mM Na₂HPO₄7H₂O/1.4 mM K₂HPO₄pH 7.4) in thepresence of phenylmethylsulfonylfluorride (PMSF, 10 μg). The homogenatewas centrifuged at 12000×g for 30 min at 4° C. and the supernatantcontaining the soluble fraction was recovered. The growth medium wasconcentrated by freeze drying and lyophilization and resuspended in 1/30vol. in PBS pH 7.4. Protein concentration was determined using theBradford reagent according to manufacturer's instructions (BioRad,Richmond, Calif.). Concentrated media samples containing 40 μg proteinand soluble extracts (50 μg protein) were subjected to 0.1% SDS/7% PAGE,Laenmnli, U. K., Nature, 227, 680-685 (1970). Purified lactoferrin (250ng, Sigma, St. Louis, Mo.) was used as standard (hLF std). The resolvedproteins were transferred to nitrocellulose filters electrophoreticallyusing the Western blot procedure, Towbin, H., et al., Proc. Natl. Acad.Sci. USA, 76, 4350-4354 (1979). Filters were blocked with Tris-bufferedsaline (TBS, 0.05 M Tris/0.15 M NaCl pH 7.5) containing 2% dried milkand then incubated by 2 h in the same with the addition of a 1 μg/ml ofa specific polyclonal IgG directed against hLF (Sigma, St. Louis, Mo.).Filter washes (5×10 min) were in TBS/0.05% Nonidet P40 followed byincubation with 1 μCi of [¹²⁵I] protein A in BS/2% dried milk. Thefilter was washed (5×10 min) with TBS/0.05% Nonidet P-40, dried andexposed overnight to Kodak XAR5 film at −70° C. The film was thendeveloped by autoradiography. The autoradiographs demonstrate productionof hLF. Western analysis was performed to determine if the hLF cDNA wasexpressed in the A. nidulans transformats under the control of the alcApromoter.

Conidia (1×10⁶/ml) from transformat No. 5, which contained the highestnumber of copies of integrated hLF cDNAs, and from untransformed GR5were inoculated into minimal medium utilizing glucose as the carbonsource. After 18 h, the cultures were harvested, washed and reinoculatedinto minimal medium supplemented with 1.2% ethanol and grown for anadditional 12 or 24 h before harvesting the cultures. Cell extracts andsamples of the growth medium were resolved by SDS-PAGE, transferred tonitrocellulose and immunoblotted using a specific polyclonal IgGdirected against hLF. An immunoreactive band indistinguishable fromnative hLF was evident in the cells and growth medium from transformatNo. 5 after 12 and 24 h growth only after ethanol induction. Cellextracts or growth medium obtained from untransformed GR5 did notcontain an immunoreactive band even after addition of ethanol. Theseresults demonstrate that hLF is expressed in transformed A. nidulansunder the control of the alcA promoter.

Western analysis revealed hLF in the cells in all of the remainingtransformants. In general there was a correlation between the plasmidcopy number and the expression levels obtained. In the medium hLF wasdetected only with transformats containing multiple copies of integratedexpressed plasmid (Nos. 1, 5, 7 and 10).

In order to monitor the levels of hLF produced in the system, a pilotfermentation of transformant No. 5 was carried out using the growthparameters described above. ELISA analysis, Vilja, P., et al., J.Immunol. Methods, 76, 73-83 (1985), using a specific biotinylated IgGdirected against hLF demonstrated that the total level of recombinanthLF produced was 5 μg/ml with approx. 30% (1.5-2.0 μg/ml) of thismaterial secreted into the medium.

Iron Binding Analysis of hLF.

To test if recombinant lactoferrin synthesized and secreted in A.nidulans has an iron binding capacity similar to authentic humanlactoferrin, samples of the growth medium of transformant No. 5 anduntransformed GR5 spores were examined using an ⁵⁹Fe microfilter-bindingassay to detect ⁵⁹Fe-bound lactoferrin. Iron-binding (⁵⁹Fe) is detectedin the medium from transformant No. 5 but not in the medium from controluntransformed GR5 spores. These results indicate that hLF produced in A.nidulans is biologically active in its capacity to bind ⁵⁹Fe.

The data demonstrate the successful production of biologically activehLF in A. nidulans. The levels of hLF produced in A. nidulans wereapprox. 5 μg/ml with 30% of the hFL secreted into the growth medium. Thesecreted hLF was identical to native breast milk LF with regard to sizeand immunoreactivity. Furthermore, the hLF was capable of binding iron.Although hLF has been reported to contain anti-fungal properties,neither the re-hLF nor native hLF when added to the growth medium,retarded the growth of this strain of A. nidulans. The production ofbiologically active hLF in A. nidulans will facilitate testing ofpossible nutritional and therapeutic uses of this protein.

EXAMPLE 16 Production of DNA Sequence Substitution Analogs

FIG. 18 shows the restriction enzyme cleavage sites in the SEQ I. D. No.1 cDNA for cleavage by various endonucleases. Table 2 lists thealternative codons that code for the 20 common amino acids. DNA sequencesubstitution analogs that also code for human lactoferrin can beconstructed by choosing alternate codons from Table 2.to alter the DNASequence between a pair of cleavage sites selected from FIG. 18.Alternative codons are assembled into a synthetic oligonucleotide byconventional methods and the synthetic oligo is substituted into theendonuclease treated DNA of Sequence ID. No. 1 by the methods describedin “Molecular Cloning. A Laboratory Manual”, 2d Edition, Cold SpringHarbor Laboratory Press (1989), to produce a substitution analog. Othermethods generally known to those skilled in the art can also be employedto obtain substitution analogs of DNA sequences. The alteration of theDNA by cleavage and codon substitution maybe repeated to substitutesubstantial portions of the original DNA sequence with alternativecodons without altering the protein expressed by the DNA of Sequence ID.No. 1. The same methods can of course be used to make substitutionanalogs of the cDNA of SEQ ID No. 3 and 5. Alteration of a DNA sequencewhich produces no change in the protein expressed by the DNA sequencemight, for example, be conducted to increase protein expression in aparticular host cell by increasing the occurrence of codons thatcorrespond to amino acid tRNAs found in higher concentration in the hostcell. Such altered DNA sequences for substitution analogs can be easilyproduced by those of ordinary skill in the art following the method setout above, or other alternative techniques for altering the DNA sequencewhile obtaining the same protein on expression. Substitution analogs canbe obtained by substitution of oligonucleotides at restriction cleavagesites as described above, or by other equivalent methods that change thecodons while preserving the amino acid sequence of the expressedprotein. TABLE 2 AMINO ACID CODONS Phe TTT TCC Leu TTA TTG CTT CTC CTACTG Ile ATT ATC ATA Met ATG Val GTT GTC GTA GTG Ser TCT TCC TCA TCG AGTAGC Pro CCT CCC CCA CCG Thr ACT ACC ACA ACG Ala GCT GCC GCA GCG Tyr TATTAC Gly GGT GGC GGA GGG His CAT CAC Gln CAA CAG Asn AT AAC Lys AAA AAGAsp GAT GAC Glu GAA GAG Cys TGT TGC Trp TGG Arg CGT CGC CGA CGG AGA AGGTERMINATION TAA SIGNALS TAG TGA

In conclusion, it is seen that the present invention and the embodimentsdisclosed herein are well adapted to carry out the objectives and obtainthe end set forth in this application. Certain changes can be made inthe method and apparatus without parting from the spirit and scopes ofthis invention. It is realized that changes are possible and that it isfurther intended that each element or step presided in any of the filingclaims is to be understood as to referring to all equivalent elements orsteps for accomplishing the essentially the same results insubstantially the same or equivalent manner. It is intended to cover theinvention broadly in whatever form its principles may be utilized. Thepresent invention, therefore, is well adapted to carry out the objectsand obtain the ends and advantages mentioned, as well as others inherenttherein.

1-23. (Canceled)
 24. A process for producing lactoferrin which comprisesculturing a transformant eucaryotic cell containing a recombinantplasmid, said plasmid comprising a plasmic vector having apolydeoxyribonucleotide which codes for a lactoferrin proteins in asuitable nutrient medium until the lactoferrin protein is formed andisolating the lactoferin protein. 25-29. (Canceled)
 30. A method forproducing biologically active recombinant lactoferrin comprising thesteps of: combining sequences containing a selectable marker gene, apromotor, a transcription termination sequence, and a linker sequence;cloning said sequences to form a plasmid; digesting said plasmid with arestriction endonuclease; inserting a cDNA coding for human, bovine orporcine lactoferrin into a restriction site; and transforming a cellswith said plasmid to produce said recombinant lactoferrin.
 31. Themethod of claim 30, wherein said selectable marker gene is selected fromthe group consisting of pryr4, pyrG, andS, argB and trpC.
 32. Canceled33. The method of claim 30, wherein said promotor is selected from thegroup consisting of alcohol dehydrogenase, argB, α-amylase,glucoamylase, alcohol dehydrogenase and benA.
 34. The method of claim30, wherein said transcription termination sequence is selected from thegroup consisting of α-amylase, glucoamylase, alcohol dehydrogenase andbena.
 35. The method of claim 30, wherein said linker sequence isselected from the group consisting of α-amylase, glucoamylase andlactoferrin. 36-57. (Canceled)
 58. A method for producing biologicallyactive recombinant lactoferrin comprising the steps of: combiningsequences containing a selectable marker gene, a promotor, atranscription termination sequence, and a linker sequence; cloning saidsequences to form a plasmid; digesting said plasmid with a restrictionendocnuclease; inserting a substitution analog of a cDNA sequenceselected from the group consisting of SEQ. ID No. into a restrictionsite; and transforming eucaryotic cells with said plasmid expressinglactoferrin cDNA which produces said recombinant lactoferrin.
 59. Themethod of claim 58, wherein said selectable marker gene is selected fromthe group consisting of pyr4, pyrG, andS, argB and trpC.
 60. Canceled61. A recombinant lactoferrin produced by the method of claim
 58. 62.The method of claim 58, wherein said promotor is selected from the groupconsisting of alcohol dehydrogenase, argB, α-amylase, glucoamylase, andbenA.
 63. The method of claim 58, wherein said linker sequence isselected from the group consisting of α-amylase, glucoamylase, alcoholdehydrogenase and benA.
 64. The method of claim 58, wherein said linkersequence is selected from the group consisting of α-amylase,glucoamylase, and lactoferrin. 65-68. Canceled.